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Sym004, a Novel EGFR Antibody Mixture, Can Overcome Acquired

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Sym004, a Novel EGFR Antibody Mixture, Can Overcome Acquired Volume 15 Number 10 October 2013 pp. 1196–1206 1196 www.neoplasia.com Sym004, a Novel EGFR Antibody Mari Iida*, Toni M. Brand*, Megan M. Starr*, Chunrong Li*, Evan J. Huppert*, Neha Luthar*, † † † Mixture, Can Overcome Acquired Mikkel W. Pedersen , Ivan D. Horak , Michael Kragh Resistance to Cetuximab1 and Deric L. Wheeler* *Department of Human Oncology, University of Wisconsin School of Medicine and Public Health, Wisconsin Institutes for Medical Research, Madison, WI; †Symphogen A/S, Lyngby, Denmark Abstract The epidermal growth factor receptor (EGFR) is a central regulator of tumor progression in a variety of human cancers. Cetuximab is an anti-EGFR monoclonal antibody that has been approved for head and neck and colorectal cancer treatment, but many patients treated with cetuximab don’t respond or eventually acquire resistance. To determine how tumor cells acquire resistance to cetuximab, we previously developed a model of acquired resis- tance using the non–small cell lung cancer line NCI-H226. These cetuximab-resistant (CtxR) cells exhibit increased steady-state EGFR expression secondary to alterations in EGFR trafficking and degradation and, further, retained dependence on EGFR signaling for enhanced growth potential. Here, we examined Sym004, a novel mixture of antibodies directed against distinct epitopes on the extracellular domain of EGFR, as an alternative therapy for CtxR tumor cells. Sym004 treatment of CtxR clones resulted in rapid EGFR degradation, followed by robust inhibition of cell proliferation and down-regulation of several mitogen-activated protein kinase pathways. To determine whether Sym004 could have therapeutic benefit in vivo, we established de novo CtxR NCI-H226 mouse xenografts and subsequently treated CtxR tumors with Sym004. Sym004 treatment of mice harboring CtxR tumors resulted in growth delay compared to mice continued on cetuximab. Levels of total and phospho-EGFR were robustly de- creased in CtxR tumors treated with Sym004. Immunohistochemical analysis of these Sym004-treated xenograft tumors further demonstrated decreased expression of Ki67, and phospho-rpS6, as well as a modest increase in cleaved caspase-3. These results indicate that Sym004 may be an effective targeted therapy for CtxR tumors. Neoplasia (2013) 15, 1196–1206 Abbreviations: CtxR, cetuximab-resistant; CtxS, cetuximab-sensitive; EGFR, epidermal growth factor receptor; ERK1/2, extracellular signal–regulated kinases 1 and 2; HP, parental NCI-H226 cells; HNSCC, head and neck squamous cell carcinoma; i.p., intraperitoneal; MAPK, mitogen-activated protein kinase; mAb, monoclonal antibody; mCRC, metastatic colorectal cancer; NSCLC, non–small cell lung cancer; siRNA, small interfering RNA Address all correspondence to: Deric L. Wheeler, PhD, Department of Human Oncology, University of Wisconsin Comprehensive Cancer Center, 1111 Highland Avenue, WIMR 3159, Madison, WI 53705. E-mail: [email protected] 1The project described was supported, in part, by grant UL1TR000427 from the Clinical and Translational Science Award program, through the National Institutes of Health (NIH) National Center for Advancing Translational Sciences, grant RSG-10-193-01-TBG from the American Cancer Society (D.L.W.), grant 888157 from Symphogen A/S (D.L.W.), and NIH grant T32 GM08.1061-01A2 from Graduate Training in Cellular and Molecular Pathogenesis of Human Diseases (T.M.B.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Conflict of interest: D.L.W. holds a laboratory research agreement with Symphogen A/S. M.W.P., I.D.H., and M.K. are employed by Symphogen A/S. No potential conflicts of interest were disclosed by other authors. Received 5 September 2013; Revised 25 September 2013; Accepted 25 September 2013 Copyright © 2013 Neoplasia Press, Inc. All rights reserved 1522-8002/13/$25.00 DOI 10.1593/neo.131584 Neoplasia Vol. 15, No. 10, 2013 Sym004 Can Overcome Resistance to Cetuximab Iida et al. 1197 Introduction In this study, we hypothesized that cells with acquired resistance The epidermal growth factor receptor (EGFR) is a member of the to cetuximab retain growth dependency on the EGFR and would human epidermal growth factor receptor (HER) family of receptor therefore benefit from therapeutic agents that promote EGFR degra- tyrosine kinases that consists of four members: EGFR (ErbB1/HER1), dation. We found that Sym004 treatment delays the emergence of HER2/neu (ErbB2), HER3 (ErbB3), and HER4 (ErbB4). Stimulation acquired resistance to cetuximab in NCI-H226 cells both in vitro of EGFR through ligand binding promotes receptor homodimerization and in vivo. Down-regulation of several MAPK pathways was also or heterodimerization with other HER family receptors, leading to acti- observed in response to Sym004 treatment. The results presented vation of its intrinsic tyrosine kinase [1,2]. Activation of EGFR initiates herein suggest that tumors with acquired resistance to cetuximab various signaling cascades, one of which is the mitogen-activated protein can thus be successfully targeted with the novel anti-EGFR thera- kinase (MAPK) network. MAPKs belong to a group of intracellular ser- peutic Sym004. ine/threonine protein kinases that consist of various members, including the extracellular signal–regulated kinases 1 and 2 (ERK1/2) and p38 MAPKs (alpha, beta, gamma, delta). Upon activation of EGFR, the Materials and Methods MAPK pathway is initiated through the promotion of Ras binding to guanosine triphosphate (GTP), which in turn activates RAF kinases Cell Lines MAPK/extracellular signal-regulated kinases (MEK), and MAPKs. The human NSCLC cell line NCI-H226 was provided by Drs J. Minna MAPKs can subsequently activate numerous transcription factors such and A. Gazdar (University of Texas Southwestern Medical School, as c-Fos, c-Myc, and Elk-1 and numerous protein kinases such as Dallas, TX). The cells were maintained in 10% FBS in RPMI-1640 ribosomal S6 kinase 1 (RSK1) [3]. Collectively, the stimulation of the (Mediatech, Inc, Manassas, VA) with 1% penicillin and streptomycin. MAPK pathway by EGFR has been shown to greatly enhance cell The development of CtxR cells has been described previously [10]. proliferation, angiogenesis, invasion, and metastasis in cancer cells. Aberrant expression or activity of EGFR has been identified in Small Interfering RNA and Transfection many human epithelial cancers, including non–small cell lung cancer For small interfering RNAs (siRNAs), CtxR cells (HC1, HC4, and (NSCLC). Therefore, targeting EGFR has been intensely pursued HC8) were transiently transfected with siEGFR (ON-TARGETplus, over the last three decades as a cancer treatment strategy. One SMART pool #L-003114-00; Dharmacon, Lafayette, CO) using approach to inhibit the activation of EGFR is through the use of Lipofectamine RNAiMAX according to the manufacturer’s instruc- monoclonal antibodies (mAbs) that bind the extracellular domain tions (Invitrogen, Carlsbad, CA). The nontargeting siRNA (ON- of EGFR to block natural ligand binding. Cetuximab (IMC-225, TARGETplus Non-targeting Pool, #D-001810-10) was obtained Erbitux) is a human/murine chimeric mAb that was developed to from Dharmacon as a control. Cells were then lysed for analysis of bind to the extracellular domain III of EGFR. This interaction par- protein knockdown by immunoblot analysis after siRNA transfection. tially blocks the ligand-binding domain and sterically hinders the correct extended conformation of the dimerization arm on domain II [4]. The Food and Drug Administration has approved cetuximab Materials treatment for patients with metastatic colorectal cancer (mCRC) and Cetuximab (IMC-225, Erbitux) was purchased from the Univer- head and neck squamous cell carcinoma (HNSCC). However, while sity of Wisconsin Pharmacy. Sym004 was generously provided by cetuximab has shown beneficial antitumor effects in these cancers, Symphogen A/S (Lyngby, Denmark). the majority of patients who initially respond eventually acquire resistance [1,5,6]. Antibodies Numerous efforts have been undertaken to define the molecular All antibodies were purchased from commercial sources as indicated mechanisms of acquired resistance to cetuximab [7–14]. Our laboratory below: EGFR, pEGFR (Y1173), and HRP-conjugated goat anti-rabbit has established panels of cetuximab-resistant (CtxR) clones from the IgG and goat anti-mouse IgG were obtained from Santa Cruz Bio- NSCLC cell line NCI-H226 by exposing these cells in vitro to increas- technology, Inc (Dallas, TX). pEGFR (Y1045), pEGFR (Y1068), ing concentrations of cetuximab. Using this model, we have shown pERK1/2 (T202/Y204), p44/42 ERK1/2, p-rpS6 (S235/236), rpS6, that impaired EGFR internalization and degradation lead to increased p-c-RAF (S289/296/301), c-RAF, pRSK1 (S380), RSK1, p-p38 EGFR surface level expression, increased EGFR kinase activity, and (T180/Y182), and p38 were obtained from Cell Signaling Technology dependence on EGFR induced signaling pathways [7]. These data (Danvers, MA). pERK1/2 (T202/Y204/T185/Y187) was purchased suggest that EGFR remains a molecular target in this setting and that from Abcam (Cambridge, MA). α-Tubulin was purchased from new therapeutics that can initiate the rapid internalization and robust Calbiochem (Billerica, MA). degradation of EGFR may be a potent anticancer strategy. Sym004 is a novel 1:1 mixture of a pair of mAbs (992 and 1024) directed against nonoverlapping epitopes on EGFR [15,16]. Sym004 Cell Proliferation Assay inhibits cancer cell growth and
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